Journal: medRxiv

Article Title: Epigenetic Alteration of Smooth Muscle Cells Regulates Endothelin-Dependent Blood Pressure and Hypertensive Arterial Remodeling

doi: 10.1101/2024.07.09.24310178

Figure Lengend Snippet: A) Schematic of the human JMJD3 gene including gene structure, H3K27Ac, DHS, blood pressure-associated SNPs, cloned fragments for luciferase assays, and vertebrate sequence conservation obtained from UCSC genome browser. Features were used to prioritize SNPs in transcriptionally relevant regions. B) Luciferase results of the DHS1 containing rs62059712 (T-major vs. C-minor) and C) DHS2 containing rs74480102 (G-major vs. A-minor) in cultured primary human smooth muscle cells (HuSMCs). D) qPCR for JMJD3 expression in HuSMCs with CRISPR-Cas9-mediated deletion of a 450 bp region encompassing the rs62059712 within DHS1 (ΔDHS1) compared with unedited (WT) HuSMCs. E) Sequence of the rs62059712 T-major and C-minor sequences with SP1 consensus binding site underlined. F) Western blot for SP1 after affinity purification using T vs. C probes corresponding to rs62059712 SNP region incubated with human aortic SMC (HuAoSMC) nuclear lysate. G) ChIP-qPCR for Sp1 at the Jmjd3 promoter in mAoSMCs compared to IgG negative control. H) ChIP-qPCR for Sp1 at the Jmjd3 promoter in mAoSMCs serum starved or treated with TGFβ. I) Luciferase assays in mAoSMCs treated with siNTC and siSp1 siRNAs then transfected with pGL3-DHS1-T and -C constructs. J) Luciferase assays in HuSMCs transfected with DHS1-C construct and then treated with TGFβ (20 ng/ml). K) qPCR for Jmjd3 expression after NTC vs. Sp1 knockdown in mAoSMCs. Data are presented as the mean ± SEM. Results are representative of data from SMCs from 4-6 mice per group, n=3 independent experiments. Data were first analyzed for normal distribution, and if data passed the normality test, a two-tailed Student’s t test was used, *p<0.05, **p<0.01.

Article Snippet: Human aortic and bronchial smooth muscle cells were purchased from Lonza and maintained in Clonetics Smooth Muscle Growth Medium-2 (SMGM-2) supplemented with growth factors, 5% FBS, and antibiotics.

Techniques: Clone Assay, Luciferase, Sequencing, Cell Culture, Expressing, CRISPR, Binding Assay, Western Blot, Affinity Purification, Incubation, ChIP-qPCR, Negative Control, Transfection, Construct, Knockdown, Two Tailed Test

Journal: medRxiv

Article Title: Epigenetic Alteration of Smooth Muscle Cells Regulates Endothelin-Dependent Blood Pressure and Hypertensive Arterial Remodeling

doi: 10.1101/2024.07.09.24310178

Figure Lengend Snippet: A) UMAP plots showing distribution of cell populations from human scRNA-seq of human femoral artery samples (n=4 samples). B) Relative expression of JMJD3 in SMCs in human artery. C) Graphical representation of Pearson correlation of smooth muscle genes obtained from single cell RNA-seq of human arteries with heat gradient representing strength of association. D) Dot plot of JMJD3 expression in human artery SMCs separated by high versus low expression of ACTA2 and CNN1 . E) Representative image of PLA assay in mAoSMCs demonstrating interaction between Srf and Jmjd3 (red foci). At least 4 high powered fields were analyzed. Scale bar, 50 um. F) Srf ChIP-qPCR using primer sets for the Acta2 , Tagln , and Cnn1 promoters in mAoSMCs treated with NTC or siJmjd3 siRNA. Data are presented as the mean ± SEM, n=3 independent experiments. N=4 arterial samples. Experiments representative of SMCs from 4-6 mice per group. Two-tailed Student’s t-test and Pearson’s correlation coefficient were used. *p<0.05, **p<0.01, ***p<0.001.

Article Snippet: Human aortic and bronchial smooth muscle cells were purchased from Lonza and maintained in Clonetics Smooth Muscle Growth Medium-2 (SMGM-2) supplemented with growth factors, 5% FBS, and antibiotics.

Techniques: Expressing, RNA Sequencing, ChIP-qPCR, Two Tailed Test